Figure 3. DHA promoted Treg generation through TGF-βR:Smad signaling.

3A. Purified CD4⁺ T cells were activated under Th17 polarizing conditions for 4 days with DHA or Mock treatment. The fractions of Foxp3 expressing Treg cells were determined by Foxp3 intracellular staining and flow-cytometry. Means ± SD of three experiments are shown.

3B. The total numbers of Treg cells recovered from experiments described in (A) were compared. Means ± SD of three experiments are shown. (*P<0.05)

3C. Purified CD4⁺ T cells from Foxp3 reporter mice (27) were activated under Th17 polarizing conditions for 4 days with DHA treatment. Foxp3⁺ Treg cells were sorted and mixed with CFSE-labeled Foxp3⁻ naïve CD4 responder T cells (Tresp.) at different ratios. Cell mixtures were activated with soluble anti-CD3 in the presence of irradiated APC. Four days post activation, the proliferative index of responder T cell was monitored by CFSE dilution and flow-cytometry. The division index of responder T cells was determined using FlowJo software to quantitate the degree of Treg cell-mediated suppression on the proliferation of responder T cells. Means ± SD of three experiments are shown.

3D. CD4⁺ T cells were activated in the presence of varying doses of TGF-β (0–2ng/ml) with the treatments of Mock (dashed line) or DHA (solid line) for 4 days. The percentages of Foxp3⁺ Treg cells were determined by intracellular staining and flow-cytometry. Means ± SD of triplicates done in one experiment representative of three are shown (*P<0.05).

3E. CD4⁺ T cells were activated under Th17 polarizing conditions with DHA (+) or Mock (−) treatment for indicated time. The protein amounts of phosphorylated-Smad2 (p-Smad2), Smad2 and β-actin protein were detected by immunoblotting. Results are representative of three experiments. Densitometry analysis of immuno-blotting was also shown. (**P<0.01)

3F. CD4⁺ T cells were activated in the presence of 1ng/ml TGF-β with the treatment of DHA (+) or Mock (−) for indicated time. The protein amounts of phosphorylated-Smad2 (p-Smad2), Smad2 and β-actin protein were detected by immunoblotting. Results are representative of three experiments. Densitometry analysis of immuno-blotting was also shown. (**P<0.01)

3G. CD4⁺ T cells were activated in the presence of 1ng/ml TGF-β with the treatment of DHA or Mock for 4 days. Cells were simultaneously treated with TGF-βRI (ALK5) inhibitor SB525334 (+) or remain untreated (−). The fractions of Foxp3⁺ Treg cells were detected by intracellular staining and flow-cytometry. Means ± SD of three experiments are shown.

3H. CD4⁺ T cells were purified from wild-type (WT) or CD4cre-Smad2^fl/fl mice (Smad2⁻) in the presence of 1ng/ml TGF-β with the treatment of DHA or Mock for 4 days. The fractions of Foxp3⁺ Treg cells were detected by intracellular staining and flow-cytometry. Results are representative of at least three experiments. Means ± SD of three experiments are shown.